Xi Jing-Hua, Bai Fang, McGaha Rebecca, Andley Usha P
Department of Ophthalmology and Visual Sciences, Washington University School of Medicine, 660 S. Euclid Ave., Campus Box 8096, St. Louis, Missouri 63110, USA.
FASEB J. 2006 May;20(7):846-57. doi: 10.1096/fj.05-5532com.
The molecular chaperones alphaA- and alphaB-crystallins are important for cell survival and genomic stability and associate with the tubulin cytoskeleton. The mitotic spindle is abnormally assembled in a number of alphaA-/- and alphaB-/- lens epithelial cells. However, no report to date has studied the effect of alpha-crystallin expression on tubulin/microtubule assembly in lens epithelial cells. In the current work we tested the hypothesis that the absence of alphaA- and alphaB-crystallins alters microtubule assembly. Microtubules were reconstituted from freshly dissected explants of wild-type, alphaA-/-, alphaB-/-, and alpha(A/B) -/- (DKO) mouse lens epithelia and examined by electron microscopic and biochemical analyses. The wild-type microtubules were 4 mum long and approximately 25 nm wide and had a characteristic protofilament structure, but alphaB-/- microtubules were 2.5-fold longer. Microtubule-associated proteins (MAPs) extracted from microtubules by washing with salt included transketolase, alpha-enolase, and betaB2-crystallin. In DKO lens epithelial microtubules but not in wild-type, alphaA-/- or alphaB-/- microtubules, extraction of the MAPs gave very long (14-20 microm) "polyfilament" assemblies that were tightly bundled. Addition of exogenous alpha-crystallin (alphaA+ alphaB) was ineffective in preventing polyfilament formation. However, normal microtubule structure could be restored by including MAPs derived from wild-type lens epithelial cells during microtubule reconstitution. Intriguingly, these data suggest that alpha-crystallin may interact with MAPs to inhibit aggregation of microtubules in lens epithelial cells. Sedimentation analysis and 90 degrees light scattering measurements showed that alpha-crystallin suppressed tubulin assembly in vitro. Alpha-crystallin did not have a strong effect on the GTPase activity of purified tubulin. SDS-PAGE analysis showed that alpha-crystallin prevented heat-induced aggregation of tubulin, suggesting that alpha-crystallin may affect microtubule assembly by maintaining the pool of unassembled tubulin.
分子伴侣αA-和αB-晶状体蛋白对细胞存活和基因组稳定性很重要，并与微管细胞骨架相关联。在许多αA-/-和αB-/-晶状体上皮细胞中，有丝分裂纺锤体组装异常。然而，迄今为止，尚无报告研究α-晶状体蛋白表达对晶状体上皮细胞中微管蛋白/微管组装的影响。在当前工作中，我们测试了以下假设：αA-和αB-晶状体蛋白的缺失会改变微管组装。从野生型、αA-/-、αB-/-和α(A/B)-/-（双敲除，DKO）小鼠晶状体上皮的新鲜解剖外植体中重构微管，并通过电子显微镜和生化分析进行检查。野生型微管长4μm，宽约25nm，具有特征性的原纤维结构，但αB-/-微管长2.5倍。通过用盐洗涤从微管中提取的微管相关蛋白（MAPs）包括转酮醇酶、α-烯醇酶和βB2-晶状体蛋白。在DKO晶状体上皮微管中，而不是在野生型、αA-/-或αB-/-微管中，提取MAPs会产生非常长（14-20μm）的紧密束状“多丝”组装体。添加外源性α-晶状体蛋白（αA+αB）对防止多丝形成无效。然而，在微管重构过程中加入源自野生型晶状体上皮细胞的MAPs可以恢复正常的微管结构。有趣的是，这些数据表明α-晶状体蛋白可能与MAPs相互作用以抑制晶状体上皮细胞中微管的聚集。沉降分析和90度光散射测量表明，α-晶状体蛋白在体外抑制微管蛋白组装。α-晶状体蛋白对纯化微管蛋白的GTPase活性没有强烈影响。SDS-PAGE分析表明，α-晶状体蛋白可防止热诱导的微管蛋白聚集，这表明α-晶状体蛋白可能通过维持未组装微管蛋白池来影响微管组装。
